过表达棉花葡萄糖醛酸激酶基因GbGlcAK促进拟南芥细胞伸长

doi:10.13304/j.nykjdb.2021.0806

摘要/Abstract

摘要：

纤维品质改良一直是棉花育种的重要目标。基于纤维不同发育时期的RNA-seq数据，发现1个在海岛棉和陆地棉间差异表达的葡萄糖醛酸激酶基因（glucuronic acid kinase/glucuronokinase，GlcAK）。从海岛棉（Gossypium barbadense）Pima90-53纤维中克隆了GbGlcAK，其ORF为1 101 bp，编码366个氨基酸。GbGlcAK具有GHMP 激酶 N和C结构域，为GHMP超家族成员，属于可溶性非分泌蛋白，与拟南芥GlcAK亲缘关系最近。亚细胞定位结果显示GbGlcAK分布在细胞质中。超表达GbGlcAK的拟南芥叶表皮毛、下胚轴、下胚轴细胞和根的长度较野生型均极显著增加，UDP-D-GlcA代谢相关基因表达量增加，果胶含量显著增加，说明过表达GbGlcAK能够上调UDP-D-GlcA代谢通路相关基因的表达从而提高果胶含量，进而促进细胞伸长，这为进一步研究GbGlcAK在棉纤维发育中的功能提供了参考。

关键词: 棉花, 葡萄糖醛酸激酶, 纤维发育, 细胞伸长

Abstract:

Fiber quality improvement is an important objective for cotton breeding. A differentially expressed gene GlcAK （glucuronic acid kinase/glucuronokinase） between sea island and upland cotton was identified based on RNA-seq data from different stages of fiber development. In this study， GbGlcAK （Gossypium barbadense GlcAK） with 1 101 bp of ORF was cloned from the fibers of sea island cotton Pima 90-53， which encoded 366 amino acids. GbGlcAK had the structure domain of GHMP kinases N and C domain and belonged to the GHMP superfamily. GbGlcAK was a soluble and non-secretory protein. In terms of phyletic evolution， GbGlcAK was most closely related to GlcAK from A.thaliana. Subcellular localization results showed that GbGlcAK was distributed in the cytoplasm. Compared with the wild type， the transgenic A.thaliana lines possessed significantly longer leaf trichomes， hypocotyls， hypocotyl cells and roots. The expression of uridine diphosphate-D-glucuronic acid （UDP-D-GlcA） metabolic related genes were upregulated， and the pectin content was also significantly increased in the transgenic lines. These results suggested that overexpression of GbGlcAK in A. thaliana up-regulated UDP-D-GlcA metabolic pathway， thereby increased pectin content and promoted cell elongation. Our study provided references for further exposing the function of GbGlcAK in cotton fiber development.

Key words: cotton, glucuronokinase, fiber development, cell elongation

中图分类号:

S562

吴楠, 杨君, 张艳, 孙正文, 张冬梅, 李丽花, 吴金华, 马峙英, 王省芬. 过表达棉花葡萄糖醛酸激酶基因GbGlcAK促进拟南芥细胞伸长[J]. 中国农业科技导报, 2022, 24(6): 36-46.

Nan WU, Jun YANG, Yan ZHANG, Zhengwen SUN, Dongmei ZHANG, Lihua LI, Jinhua WU, Zhiying MA, Xingfen WANG. Overexpression of a Cotton Glucuronokinase Gene GbGlcAK Promotes Cell Elongation in Arabidopsis thaliana[J]. Journal of Agricultural Science and Technology, 2022, 24(6): 36-46.

图/表 9

表1 试验中用到的PCR和qPCR引物

Table 1 PCR and qPCR primers used in this study

引物名称 Primer names	登录号 Accession No.	引物序列 Primer sequences （5’-3’）	用途 Purpose
GlcAK-F	—	TGGTATTGGCTTGGTGCAGT	基因克隆 Gene cloning
GlcAK-R	—	CAAAAACAATCCCTAAAATGCTCTT	基因克隆 Gene cloning
sGbGlcAK-R	—	GGTGGTACCCTGCTTAGACAATGT	亚细胞定位Subcellular location
GbGlcAK-F	—	GGCGTCTAGAATGGATCAAAATATG	亚细胞定位和载体构建 Subcellular location and vector construction
GbGlcAK-R	—	GGTACCCGCCTACTTAGACAATG	载体构建 Vector construction
qGbGlcAK-F	—	CATCATCAACCCTCACCCCATTC	拟南芥实时定量PCR Real-time PCR for A. thaliana
qGbGlcAK-R	—	GCCGCATACAATAGCACTGGACC
qAtGlcAK-F	AT3G01640	GGATAGAGCATCGGTCCTTCGC
qAtGlcAK-R	AT3G01640	CGCCATTAGCAACCTTACCCCA
qAtMIPS-F	AT4G39800	AAGGAGAAGAATAAGGTGGATAAGGTT
qAtMIPS-R	AT4G39800	GCAATCGCATAAAGTGTTGAAGG
qAtIMP-F	AT3G02870	GACAGAGACTGATAAAGGATGTGAAGA
qAtIMP-R	AT3G02870	AGGGAACCCGTGAACGAAAT
qAtMIOX-F	AT4G26260	CGAAGCCATCCGCAAAGATTACC
qAtMIOX-R	AT4G26260	CCAACAACAGCCCATTGAGGAAGTC
qAtPGM-F	AT1G23190	GCTACCTACGGTCGTCACTATTACACTCG
qAtPGM-R	AT1G23190	GTAACGGATTCCCTGGTGCTTCG
qAtUGP-F	AT5G17310	GCCCAGCAAGGGAAAGACCG
qAtUGP-R	AT5G17310	CGATGGCACCCAAGTTGTCTGAAT
qAtUGD-F	AT5G15490	GATTGCGGTTCTCGGCTTCG
qAtUGD-R	AT5G15490	TGCTTCACAGTGGTGGGGCTC
qAtUXS-F	AT5G59290	TGAGAAGAATGAGGTGGTTGTTGC
qAtUXS-R	AT5G59290	GGTTGTATTTGTAGAAGATAGGAGAGGC
qAtGAE-F	AT3G23820	GCGACGGCGGATACAAGCA
qAtGAE-R	AT3G23820	GATGGCGAAGATGAGGACGAGG
qAtUB5-F	AT3G62250	CCTCGCCGACTACAACATCCAG
qAtUB5-R	AT3G62250	CTTCTTCCTCTTCTTAGCACCACCA

图1 GbGlcAK蛋白特征A：ProtScale分析GbGlcAK肽链亲/疏水性分布曲线；B、C：TOPpred2和TMHMM 2.0工具对GbGlcAK氨基酸序列的跨膜区预测；D：GbGlcAK蛋白信号肽分析

Fig. 1 Characterization of GbGlcAK proteinA： Hydrophilic/hydrophobic distribution curve of the GbGlcAK peptide analyzed by ProtScale； B， C： Transmembrane region prediction of the GbGlcAK amino acid sequence by TOPpred2 and TMHMM 2.0； D： Signal peptide analysis of the GbGlcAK protein

图2 GbGlcAK系统进化分析注：Rc—蓖麻；Pt—毛果杨；Vv—葡萄；At—拟南芥；Gm—大豆；Os—水稻；Zm—玉米；Sb—高粱。

Fig.2 Evolutionary analysis of GbGlcAKNote： Rc—Ricinus communis； Pt—Populus trichocarpa； Vv—Vitis vinifera； At—Arabidopsis thaliana； Gm—Glycine max； Os—Oryza sativa； Zm—Zea mays； Sb—Sorghum bicolor.

图3 GbGlcAK的亚细胞定位A、B、C：蓝光、可见光叠加蓝光、可见光下的转pCam：：GFP细胞；D、E、F：质壁分离后蓝光、可见光叠加蓝光、可见光下的转pCam：：GbGlcAK-GFP细胞

Fig. 3 Subcellular localization of GbGlcAKA， B， C： Cell transfected with pCam：：GFP under Blu-ray， visible light superimposed Blu-ray， and visible light； D， E， F： Cell transfected with pCam：：GbGlcAK-GFP under Blu-ray， visible light superimposed Blu-ray， and visible light after plasma-wall separation

图4 转基因拟南芥叶表皮毛长度分析注：**表示在P<0.01水平差异显著（双尾t检验）。

Fig. 4 Analysis of leaf trichome length in transgenic A. thalianaNote：** indicates significant difference at the P<0.01 level （2-tailed t-test）.

图5 转基因拟南芥下胚轴长度分析A、B：超表达GbGlcAK拟南芥与野生型拟南芥下胚轴长度比较；C：显微镜下观察超表达GbGlcAK拟南芥与野生型拟南芥叶下胚轴细胞。**表示P<0.01水平的显著性（双尾t检验）；白色箭头所示细胞边界

Fig. 5 Analysis of hypocotyl length in transgenic A. thalianaA， B： Comparison of hypocotyl length between transgenic and wild-type Arabidopsis； C： Microscopic observation of hypocotyl cells； ** indicates significant difference at the P<0.01 level （2-tailed t-test）；white arrows show the ends of cells

图6 转基因拟南芥根长分析注：**表示P<0.01水平的显著性（双尾t检验）。

Fig. 6 Analysis of root length in transgenic A. thalianaNote：** indicates significant difference at the P<0.01 level （2-tailed t-test）.

图7 转基因拟南芥中UDP-D-GlcA代谢相关基因的表达分析

Fig.7 Expression analyses of UDP-D-GlcA metabolism related genes in transgenic Arabidopsis

图8 转基因拟南芥果胶含量注：*表示P<0.05水平的显著性（双尾t检验）。

Fig. 8 Pectin content in transgenic ArabidopsisNote：* indicates significant difference at P<0.05 level （2-tailed t-test）.

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